Biophysical characterization and solution structure of the cannulae forming protein CanA from the hyperthermophilic archaeon Pyrodictium abyssi

CanA from Pyrodictium abyssi , the main constituent of the extracellular protein network of this archaeon, forms a hollow-fiber network in the presence of divalent ions. A non-polymerizing mutant, K ₁ -CanA, was created. The apparent hydrodynamic radius is 1.68 and 1.74 nm, as determined by NMR diffusion measurements and dynamic light scattering. As shown by transmission electron microscopy, cannulae produced in vitro from the polymerizing recombinant protein, have the same form and dimension (diameter of 30 nm) as natural cannulae. The solution structure of CanA was determined by multidimensional NMR spectroscopy. It mainly consists of β-pleated sheets and 2 small α-helices, arranged as β ₁ β ₂ β ₃ β ₄ α ₁ β ₅ β ₆ α ₂ β ₇ β ₈ β ₉ β ₁₀ β ₁₁ β ₁₂ β ₁₃ . Of the 13 β-strands, 8 form a non-canonical Jelly Roll Barrel class I fold. Several interaction sites for divalent ions could be identified by [ ¹ H, ¹⁵ N]-SOFAST-HMQC spectroscopy located at the two ends of the Jelly Roll Barrel. In binding area 1, specific Ca ²⁺ -sites with KD-values of 0.77 ± 0.05 mM are observed.